production of ultrapure EPA and polar lipids form largely heterotrophic culture

ABSTRACT

Eicosapentaenoic acid (EPA) compositions and EPA-rich polar lipids for prophylactic or therapeutic applications are described. Production from certain cultured micro-organisms (like  Nitzschia laevis ) promotes synthesis of EPA, including polar lipids including EPA. The EPA-rich polar lipids themselves may be used as polar compounds. EPA can be selectively hydrolyzed from particular positions in isolated polar lipids by lipase activity, then optionally further purified. The process bypasses reliance on diminishing fish stocks and on physico-chemical processes that may not adequately separate desirable n-3 HUFAs from unwanted products like DHA also found in fish oil and cultured organisms.

CROSS REFERENCE OF RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 12/307,532, filed Nov. 5, 2009, and entitled “PRODUCTION OFULTRAPURE EPA AND POLAR LIPIDS FROM LARGELY HETEROTROPHIC CULTURE,”which is a National Phase of International Patent Application No.PCT/NZ2007/000172, filed Jul. 5, 2007, which claims the benefit of NewZealand Patent Application No. 548339, filed Jul. 5, 2006, all of whichare incorporated by reference herein in their entireties.

FIELD OF THE INVENTION

This invention relates to lipid compositions synthesized by single-cellorganisms, to the manufacture and some applications of omega-3 highlyunsaturated fatty acids and to pharmaceutical substances, and inparticular to novel therapeutic, dietary and health-food compositions.

DEFINITIONS

Alpha linolenic acid is an omega-3 fatty acid with eighteen carbon atomsand three double bonds.

Arachidonic acid is an omega-6 fatty acid with twenty carbon atoms andfour double bonds.

Diacyl galactolipids are galactolipids with both Sn1 and Sn2 occupied byfatty acid molecules.

Digalactosyl galactolipids are those where two galactose molecules areattached.

Docosopentaenoic acid is an omega-3 fatty acid with twenty-two carbonatoms and five double bonds.

Docosahexanoic acid is an omega-3 fatty acid with twenty-two carbonatoms and six double bonds.

Eicosapentaenoic acid is an omega-3 fatty acid with twenty carbon atomsand five double bonds.

Effective amount means: an amount sufficient to cause a desired resultwhen administered.

EPA-only means: a composition containing in its highly unsaturatedomega-3 fatty acid component, substantially only EPA.

Esterification of n-3 HUFAs is understood, so that a term such as “99%pure EPA” is understood to take no account of the ethyl or other moietyused to form an ester with the free fatty acid.

Nutraceutical is any substance that is a food or a part of a food andprovides medical or health benefits, including the prevention andtreatment of disease.

Purity: 1. Chemical purity: The EPA composition is substantially free ofall molecules other than EPA.

2. Functional purity: The EPA composition is substantially free of thosematerials herein defined as “undesired”. An EPA composition according tothis definition of purity may include only 1 to 10% EPA (although morepreferably 60-90% EPA; the remainder may include galactolipids orcomponents thereof, or added pharmaceutically acceptable excipients,anti-oxidants, adsorbents, flavours, and the like.

3. Pharmaceutical purity: refers to EPA-rich compositions comprisingaround 90% of EPA or more by weight and simultaneously a ratio of EPA toany individual undesired molecule of at least around 90 to 1. Undesiredmolecules or materials are defined as those that may diminish thedesired beneficial health effect of EPA-rich compositions whenco-consumed. These include molecules that may diminish the desiredeffect through actions which may be antagonistic, competitive, block,reverse, mediate, synergise or otherwise alter the desired beneficialhealth effect of EPA when consumed in an effective (or clinicallyrelevant) amount. For the purposes of the present specificationundesired molecules are to include structurally or functionally similarmolecules including fatty acids, docosohexae{acute over (η)}oic acid(DHA), arachidonic acid (AA), 18:4 n-3, 18:3 n-3, 18:2 n-6 and otheromega-3 and 6 fatty acids in general. AA is a precursor of EPA.

EPA-rich means that the composition includes more than 1% of EPA as dryweight.

EPA productivity means the amount of EPA which can be produced per unitcost. A proxy measure for productivity in largely heterotrophic culturesof microalgae culture is often taken as the combined effect of yield,growth rate, cell density, nutrient utilisation efficiency, and dilutionrate.

Fatty acid compositions include—ethyl esters, salts, free fatty acids,methyl esters, and other alcohol esters of fatty acids, andcombinatorial lipids.

Galactolipids are comprised of a glycerol backbone with at least twoseparate molecules attached; at least one of which is a fatty acid andthe other is. either one or two molecules of galactose (in mono ordisaccharide form) bonded by, in the case of the fatty acid or fattyacids, an ester link or rarely an ether link and in the case of thegalactose or galactoses an ether link in all cases. At least onegalactose is present and normally attached to the Sn3 position ofglycerol. The galactose is always in pyranose form. A second and rarelya third galactose molecule is attached by beta-d-3-pyranosyl bonds tothe preceding galactose molecule. Positions are labeled according to aconvention based on an original stereospecific structure (see table 1showing the 1.3 isomer of the DGMG galactolipid class containing an EPAmolecule' acylated in the Sn1. Normally when only one fatty acidmolecule is attached it occupies the Sn1 position. However, occasionallyone fatty acid may be attached in the Sn2 position while the Sn1position remains unoccupied. This may be referred to as a lysoderivative.

The 1,3 Isomer of DGMG, with an EPA Molecule Acylated to the Sn1Position.

Gamma linolenic acid is an omega-6 fatty acid with eighteen carbon atomsand four double bonds.

Heterotrophic culture means a culture of organisms for which the soleenergy source is derived from supplied nutrients (the major nutrientgroup for energy metabolism) which is usually a form or forms of organiccarbon (e.g. glucose, acetate).

Largely (or partially) heterotrophic culture means a mixotrophic cultureof organisms for which the major energy source is derived from suppliednutrients (the major nutrient group for energy metabolism) which isusually a form or forms of organic carbon (e.g. glucose, acetate) andthe minor energy source for energy metabolism is light.

Linoleic acid is an omega-6 fatty acid with eighteen carbon atoms andtwo double bonds.

Monoacyl galactolipids are galactolipids with only one position occupiedby a fatty acid. In a monoacyl galactolipid the fatty acid molecule isattached either at position Sn1 or Sn2.

Monogalactosyl galactolipids are those with only one galactose moleculeattached.

Neutral lipids are those lipids contained in an organism which can beisolated through the use of non-polar solvents and include mono- di- andtriacylglycerols

Photosynthetic lipids are those polar lipids whose production can besignificantly altered via the addition of light to a largelyheterotrphic culture of microorganisms. These may occur within thechloroplast as is predominantly the case with galactolipids or may alsobe associated with other cell organelles as is predominantly the casewith phospholipids.

Polar lipids are those lipids contained in an organism which can beisolated through the use of polar solvents and include phospholipids andgalactolipids

Photosynthetically active average irradiance inside a culture is theamount of electromagnetic radiation between wavelengths 400 nm and 700nm incident on a culture averaged over all positions in the culturevessel and over time.

Stearidonic acid is an omega-3 fatty acid with eighteen carbon atoms andfour double bonds.

Sub-photosynthetic exposure means an exposure to light whereby thecombination of intensity and exposure time is equal or less than aroundthe equivalent of a continuous illumination of 1 to about 10 micromolphotons per square meter per second.

Omega-3 fatty acid is a fatty acid with the first double bond threecarbon atoms from the n-methyl end of the molecule.

Omega-6 fatty acid is a fatty acid with the first double bond six carbonatoms from the n-methyl end of the molecule.

Abbreviations

18:2 n-6 or LA: linoleic acid

18:3 n-3 or ALA: alpha linolenic acid

18:4 n-3 or SDA: stearidonic acid

18:4 n-6 or GLA: gamma linolenic acid

20:4 n-6 or AA: arachidonic acid

20:5 n-3 or EPA: eicosapentaenoic acid

22:5 n-3 or DPA: docosopentaenoic acid

22:6 n-3 or DHA: docosohexaenoic acid

DGDG digalactosyldiacylgalactolipid

DGMG: digalactosylmonoacylgalactolipid

LPC: Lysophosphatidylcholine

n-3: omega-3

n-6: omega-6

n-3 HUFA: omega-3 highly unsaturated fatty acid

MGMG: monogalatosylmonoacylgalactolipid

MGDG monogalactosyldiacylgalactolipid

N. laevis: Nitzschia laevis (UTEX 2047)

N. alba: Nitzschia alba

PC: Phosphatidylcholine

LPC: Lysophosphatidylcholine

PG: Phosphatidylglycerol

PE: Phosphatidylethanolamine

SQDG: sulfoquinovosyl diacylglycerol

BACKGROUND

It was discovered in the late 1920s that certain “essential” dietaryfatty acids must be present in effective quantities for normal growthand health in rats to ensue (Burr & Burr J. Biol. Chem., 82: 345-3671929). Epidemiological data collected from human populations beginningin the 1940s then suggested relatively high dietary intakes of n-3 HUFAmay be protective against the development of a number of medicalconditions and that low n-3 intake may increase risk (Sinclair. Lancet1:381-3 1956; Bang et al., Lancet 1:1143-5.1971; Hirai of al., Lancet2:1132-3. 1980; Kromhout of al Am J. Clin. Nutr., 85:1142-1147).

In recent decades supplementation studies incorporating individualomega-3 highly unsaturated fatty acids (n-3 HUFA) in the diet of humanshave demonstrated beneficial health effects of individual dietary n-3HUFA. In particular, human dietary supplementation studies incorporatingrelatively pure forms of the n-3 HUFA eicosapentaenoic acid (EPA) havesuggested this nutrient may promote health and ameliorate or evenreverse the effects of a range of common diseases, including but notlimited to certain forms of cardiovascular disease and depression(Yokoyama et al., Lancet 369:1062-1063. 2007; Peet & Horrobin Arch. Gen.Psych. 59 (10) 913-9 2002).

The therapeutic effect of dietary supplementation with concentratedforms of EPA are dependent to some extent on purity. High purity doseforms have an advantage in terms of increased bioavailability.Furthermore the desired effects of EPA are limited or even reversed bythe co-consumption of undesired molecules; (as herein defined) inparticular docosohexaenoic acid (DHA); also AA and other omega-3 and 6fatty acids in general. Therefore to enable effective pharmaceutical ortherapeutic use of EPA, high purity dose forms, free of the undesiredmolecules, are required.

Should the demand for high purity EPA increase, which seems likely,large numbers of clinically or subclinically diseased persons may cometo depend on continuity of supply long term to maintain quality of life.To date, however, commercial manufacturers have not been capable ofeconomically producing EPA-only compositions with relatively high EPApurities which are at the same time devoid of undesired molecules.

Reasons include: (1) The raw material for commercial production isexclusively limited to particular fish oils containing high levels ofundesired molecules. (2) The undesired molecules contained in fish oilare structurally or physico-chemically similar to EPA and cannot beeasily removed during purification (3) The cost of further purificationrises in a non-linear fashion with increasing purity.

Consequently even at EPA purities up to around the high 90th percentileup to 1% or more of these undesired molecules may remain.

Purification processes are also rendered less efficient by therelatively complex mixture of fatty acids, and a high degree of naturalvariability contained in fish oil.

The practical effect of the abovementioned factors is that commercialproducts currently available that contain high purity EPA may alsocontain unacceptably high concentrations of the above-mentionedundesired molecules for therapeutic use. Furthermore the high cost ofpurifying fish oil to an extent where only small amounts of undesiredmolecules remain constrain the use of these ultrapure compositions.

Up to 15 kgs of high EPA fish oil are required to produce 1 kg of highlypurified EPA in current purification processes. Because the efficiencyof such manufacturers is sensitive to the initial concentration of EPAthese are based on fish caught with a high percentage of EPA in theirlipids. The fish oil must also be carefully handled and stored duringprocessing to protect against damage which can result in the formationof unacceptable molecular species such as trans EPA which is anunacceptable contaminant in therapeutic formulations and virtuallyimpossible to remove during purification. The complex structure of thefishing industry, the careful handling requirements and the dwindlingand finite resource of high EPA fish species means that production ofhigh purity EPA from sea fish is difficult to scale up in order to meetincreasing demands and is likely to be unsustainable.

Many publications have reported the potential of alternative sources ofEPA-rich compositions or EPA produced from cultured microbes including(micro)algae, fungi, and bacteria. Some of these sources contain lowlevels of undesired fatty acids. Additionally, the generally lesscomplex fatty acid composition of microbes as compared to fish oil mayoffer advantages in purification. Variation in fatty acid composition incultured microorganisms is minimal as compared to fish oil conferring anadditional advantage for purification. Production of EPA-richcompositions in biotechnological processes is likely to be rapidlyscalable and provide EPA-rich compositions suitable for both nutritionaland therapeutic use that are of consistent quality.

The majority of the publications relating to production of EPA frommicroalgae concern the development of outdoor production systems. Theadvantage of these systems is the main source of energy forgrowth,-sunlight, is free. Outdoor production systems however sufferfrom several key defficiencies. Firstly contamination from competingmicroorganisms limits the applicability of open pond or raceway culturesto species which are able to withstand environmental conditions thatlimit the growth of other competing microorganisms. Secondly“photobioreactor” production systems designed to restrict contaminationrequire very large surface to volume ratios to facilitate penetration oflight into the culture creating a requirement for large upfront capitalexpenditure in the establishment of these systems and an ongoingtechnical challenge and cost with regard to maintaining sterility.

A further weakness of largely photosynthetic cultures developed to dateis that species have not yet been isolated that accumulate significantquantities of intracellular lipid in the form of triglycerides whenproduced photosynthetically. This limits EPA production to thataccumulated in polar lipids, the upper limit of which appears to beunder tight physiological regulation.

Mixotrophic production systems have been proposed for production ofEPA-rich microorganisms. These provide a proportion of the energy forgrowth in the form of organic carbon supplied to the culture medium. Anadvantage of mixotrophy includes higher productivities than areachiecable with solely photosynthetic production and potentially alsolower the overall requirement for light. A disadvantage of the additionof organic carbon sources to outdoor photobioreactor cultures however isthe creation of an additional contamination risk by presenting asubstrate for growth of non-photosynthetic contaminating organisms.

A number of solely heterotrophic systems for producting EPA-richmicroorganisms have been disclosed. These overcome many of thelimitations of photosynthetic systems due to their ability to achievegrowth of EPA rich species in the absence of light. By eliminating therequirement for light it is possible to significantly reduced thesurface-to-volume ratio of reactors and consequently also reduce capitalexpenditure and sterilisation costs. An additional advantage ofheterotrophic production systems is that culture parameters can betightly controlled leading to production of a product of a consistentquality.

Lipid Classes and Fatty Acid Profile

The fatty acid composition of certain EPA-rich microalgae contain lowproportions of fatty acids with structural similarity to EPA. Togetherwith the generally less complex fatty acid composition of microalgaethis may offer advantages in terms of purification over fish oil.

In addition to achieving a favorable overall fatty acid composition incultured EPA-rich microalgae the selective production of EPA inparticular lipid classes is also possible.

One particular strategy for enhancement of lipid and overall EPAproduction in EPA-rich microalgal species is the timed imposition of anitrogen limitation in microbial culture medium in heterotrophiccultures of microalgae. When microorganisms are deprived of keynutrients required for synthesis of membranes, lipids may be accumulatedin the form of triglyceride, a lipid class not utilized extensively inlipid membrane structure.

EPA-rich triglycerides are of potential therapeutic value. EPA may berecovered from triglcerides and further purified via an array ofconventional and emerging techniques. Processes designed to extract,concentrate or purify EPA-rich lipid or fatty acid compositions fromtriglycerides however may be disadvantaged by the presence of arelatively high level and wide range of undesirable fatty acidmolecules, and a low level of stereospecificity in terms of the locationof EPA within the triglycerides.

Certain polar lipid classes produced in cultures of microalgae arerelatively rich in EPA. At the same time some of these lipid classes mayexhibit a high degree of stereospecificty in terms of the location ofEPA within the class and its isomers. This concentration of EPA in apredictable manner in particular lipid classes provides an additionalopportunity to sequester undesirable molecules in unused fractionsduring a purification process. In addition certain lipid classesproduced by cultures of microalgae may also have therapeutic value intheir own right.

It may seem surprising then that little, if any, attention has beengiven to the possibility of inducing heterotrophic or largelyheterotrophic cultures of microalgae to localise EPA in polar lipidreservoirs in such a way as to enhance the efficiency and applicabilityof extraction, concentration and purification processes and to provide asource of polar lipid for incorporation into therapeutic products. Infact prior art disclosures appear to teach away from this possibility.

Unfortunately until now strategies applied to enhancing the productivityof processes providing alternative sources of EPA-rich compositions haveled to a reduction in the polar lipid content of EPA-richmicroorganisms.

Microalgae produce two major types of polar lipids;—phospholipids andglycolipids. All these major polar lipid classes comprise a glycerolbackbone with three positions conventionally labeled Sn 1-3. Phospho andgalacto lipid classes are categorised respectively according tophosphate- and galactose-containing functional groups which are attachedto the glycerol backbone usually at the Sn-3 position. Fatty acids areacylated at one or more positions 1-2. Isometric forms of these lipidclasses arise from acylation patterns where not all available positionsare occupied by fatty acids or where a functional group is attached atan alternative position.

Galactolipids are produced predominantly in the chloroplast and are astructural component of the photosynthetic membrane. Galactolipids areone of the most polar of all the lipid classes; there is a substantialdifference in charge distribution over the molecule because of the polarnature of the one or more galactose moieties that are attached to theglycerol backbone, providing spacially separated centres of positive andnegative charge. Hence galactolipids have found application asemulsifying agents and have been proposed as drug delivery conjugates.

The polar nature (among other physiochemical properties) ofgalactolipids leads to a number of useful opportunities, including butwithout limitation to potential advantageous routes for extraction andpurification of galactolipids and galactolipid fatty acids, formulationof galactolipids and galactolipid fatty acids into foods, functionalfoods, beverages, pharmaceutical and industrial compositions, deliveryof galactolipids and galactolipid fatty acid nutritional and therapeuticproducts in a bioavailable form, as well as advantageous therapeuticeffects and mechanisms of action their use may promote.

Phospholipids are major structural components of cellular membranes. Thehighly polar ‘head’ of the molecules coupled with their hydrophobicfatty acid ‘tails’ lead the phospholipids to spontaneously form micellesand bilayers in aqueous media. Phosopholipids both within and externalto the chloroplast are expected to play a number of important roles inrelaton to the physiological response of microorganisms to light. Orexample it has been proposed that fatty acids located in cytoplasmicphospholipids classes are a reservoir for incorporation intochloroplastic lipids during production of photosynthetic membranes. Thepolar nature of phospholipids among other physiochemical propertiespresents a number of useful opportunities similar to those stated abovefor galactolipids. Certain phosopholipids including PC are known beabsorbed differentially in mammals which could be turned to atherapeutic advantage. Work on absorption of galactolipids in particularMGDG in mammals is limited.

PRIOR ART CULTURE

Cohen et al. Journal of Applied Phycology 5: 109-115, 1993 disclose ageneral scheme for obtaining microalgal galactolipids and producingcompositions enriched with the fatty acid GLA. The method disclosedinvolves extracting the total lipids of the organism and then separatingthe galactolipids from the total lipid fraction. These authors recognisein the same prior art publication that in order to be industriallyuseful the content of GLA in the microalgae (and presumably in thegalactolipid fraction) would have to be increased. The organisms used inthis study were grown under totally photosynthetic conditions. To ourknowledge, prior to the present invention neither Cohen and colleaguesnor any other previous authors have suggested that the requiredincreases in yield could be accomplished by using largely heterotrophicgrowth.

Kyle at al in U.S. Pat. No. 5,567,732 disclose a method for producingEPA-rich oils from cells of the diatom Nitzschia alba in the dark andteach that it is possible to induce this organism in heterotrophicculture to enter an oleogenic phase by allowing nitrogen depletion tooccur and after 12-24 hours allowing a silicate depletion state to alsooccur, while continuing to supply other nutrients to the culture. Thecolourless species of diatoms are preferred. (Colourless species ingeneral and in particular the microorganism preferred by Kyle at al arecolourless because they do not exhibit the phenotype of photosyntheticpigments. N. alba for example is believed to be an obligate heterotrophwhich means that it does not have any active photosynthetic capacity.Nevertheless, a published lipid class analysis of N. alba reports that afew percent of the lipid composition are comprised of galactolipids.)The authors claim that diatoms can successfully be economicallycultivated to produce large quantities of single cell oil and they state“for the purposes of this specification, single cell oil means atriolvceride product of a unicellular microorganism”. To our knowledgeprior to the present invention neither Kyle et al nor previous authorsto our knowledge have disclosed heterotrophic or largely processesuseful in the commercial co-production of EPA-rich polar lipids.

The mixotrophic production of the EPA-rich microalga Phaeodactylumtricornutum in a tubular photobioreactor is disclosed in Ceron Garcia etal Journal of Applied Phycology 12: 239-248, 2000. This manufactureutilizes 9.2 g L⁻¹ glycerol as an organic carbon source and supplies anexternal irradiance of 165 μmol photons m⁻²s⁻¹ to the photobioreactorsurface. These authors claim that by reducing the need for light thisform of mixotrophic growth has a number of advantages including thepossibility of greatly increasing the algal cell concentration and EPAproductivity in outdoor mass culture on a large-scale. Ceron Garcia andcolleagues did not however identify the advantages of largelyheterotrophic growth in terms of increased polar lipid production.Furthermore neither these authors nor any previous authors haveidentified the potential for utilizing relatively low levels ofirradiance in largely heterotrophic culture for producing EPA-richmicroorganisms.

A number of prior art publications have disclosed that cultureconditions including light intensity and wavelength can enhance lipidand overall EPA production in specified EPA-rich microalgal species.However, prior to the present invention it had not been proposed thatsub photosynthetic light intensities could be used to alter the relativeproduction of lipid classes in a commercially useful manner. Nor had itbeen proposed that sub photosynthetic light intensities could beutilized to alter the localization of EPA in lipid reservoirs ofmicroalgae in a commercially useful manufacture.

Extraction of Polar Lipids from Microalgae

Several techniques of potential industrial utility have been proposedfor extraction and concentration of galactolipids and/or fatty acidsfrom galactolipid fractions of biological material. Winget (U.S. Pat.No. 5,767,095) describes in detail a range of extraction andconcentration techniques used to recover particular lipid classes,including relatively pure galactolipids containing EPA, from a number ofphotosynthetically produced microalga including those of the diatomgenus Chlorella.

Cohen et al (J. Appl. Phycol. 5: 109, 1993) disclose the fatty acid DHAmay be produced from the phosphatidylethanolamine (PE) lipid fraction ofthe photosynthetic organism Isochrysis galbana by extracting totallipids and subsequently producing DHA rich compositions by employing thewell known technique of urea crystallisation. Vali et al U.S. Pat. No.6,953,849 disclose a process involving dewaxing of rice bran and hexaneextraction and includes HPLC with a silicic acid column. Colarow U.S.Pat. No. 5,284,941 discloses a method involving solvent boric acid gelseparation. Buchholz et al U.S. Pat. No. 5,440,028 discloses a methodisolation through membrane separation, with pH adjustment. Bergqvist etal 1995 report after their work on oat kernels that galactolipids may becommercially extracted from a range of biological materials using asolid phase extraction using the known differences in solubility inacetone between phospholipids and glycolipids. They started with a hotethanol extraction then used hexane then hexane/acetone then acetone.

No prior art publications are known to teach that it is possible toselectively isolate EPA-rich compositions in a commercial manufacturefrom the lipids of organisms that have been cultured using largelyheterotrophic culture capable of enhancing EPA productivity andsimultaneously increasing the concentration of EPA in specific polarlipid fractions.

Enzymatic Purification

A number of prior art publications disclose the use of enzymes toliberate lipids and arrive at concentrated and purified lipid and fattyacid containing compositions from fish oil and other starting materials.The inventors appreciate that various lipases and phospholipases arecapable of dis-assembling lipids. For example a variety of solvent-basedextraction systems and crystallisation techniques have been disclosedthat favour extraction of lipids of a particular class or fatty acids ofa particular chain length or degree of unsaturation. These enzymes whichmay include lipases and proteases are known to act preferentially ondifferent substrates. In the case of lipases for example, enzymes areexpected to some degree to be specific for lipid class, fatty acid, andthe position of the fatty acid within the lipid class. The activity andpreference of enzymes can be altered by altering environmentalconditions such as temperature, and via the addition of cofactors andtechniques such as immobilization.

A common analytical technique used to estimate the localisation of fattyacids at different positions in the lipid structure is to expose a fattyacid class to a lipase capable of selectively hydrolysing fatty acidslocated in a particular position. It follows that the common generalknowledge of those skilled in the art includes the recognition that boththe proportion of lipids and the localisation of target fatty acids aswell as co-localisation or lack thereof of undesired acids within lipidreservoirs of a biological material constitute critical aspects in apurification process at an analytical scale. To our knowledge however noprevious authors have disclosed methods of producing therapeutic orprophylactic compositions via the selective enzymatic hydrolysis ofalgal polar lipids at least not from polar lipids produced in largelyheterotrophic cultures.

Applications of Galactolipids

Winget teaches use of topically applied MGDG-EPA compositions in theprevention and treatment of inflammation, but does not discloseapplication of lipase-type or indeed any enzymes. Later, Bruno et al(Eur J Pharmacol: 524; 159-168 7 Nov. 2005) disclose that thegalactolipid classes MGDG, DGDG and SQDG obtained from thermophilicblue-green algae have in-vivo anti-inflammatory activities in acroton-oil induced mouse ear inflammatory response. However there is noindication in the abstract that any of the n-3 HUFAs were present.

OBJECT

The present invention provides novel methods for obtaining EPA-richcompositions that provide the public with a useful choice. In addition,the present invention provides compositions including EPA-richgalactolipids and highly purified EPA-rich fatty acid compositions thatprovide the public with a useful amount of therapeutic, prophylactic, ordietary EPA, or at least provides the public with a useful choice.

STATEMENT OF INVENTION

In a first broad aspect the invention provides a process for obtainingan eicosapentaenoic acid (EPA)-rich composition for therapeutic orprophylactic use, wherein the process employs a culture ofmicro-organisms of a type selected for a capability of largelyheterotrophic growth, and a capability of production of EPA, and acapability of photosynthetic lipid production; the process including aculture phase in which cells are grown under conditions in which organiccarbon is used as an energy source; the conditions including use ofcontrolled illumination at a level corresponding to an averagephotosynthetically active irradiance inside the culture of less than 40μmol photons m⁻²s⁻¹ and including imposition of limitation of nutrientsselected from a range including phosphorus and silicon; said proceduresbeing undertaken in order to maximise the amount of recoverable polarlipids including at least one EPA side chain, and a harvesting processthat creates a composition rich in EPA.

In a related aspect the invention provides a process as previouslydescribed in this section wherein the culture of micro-organismscomprises identified microalgae, funghi or bacteria.

Preferably the micro-organisms are comprised of the marine single-celleddiatom known as Nitzschia laevis, University of Texas microalgalcollection UTEX 2047.

In another related aspect, the micro-organisms comprises a strain ofmicro-organism selected, when under culture conditions, for an improvedyield of recoverable polar lipids having molecules which include atleast one side chain bearing EPA.

Preferably the micro-organisms accumulate galactolipids rich in EPAconcentrated at the Sn1 position within the galactolipid or likeclasses.

More preferably the micro-organisms are capable of accumulatingcommercially useful quantities of polar lipids, including galactolipidsrich in EPA at the same time as exhibiting high EPA productivity ingeneral, including the EPA found in triglycerides.

In particular, the EPA-rich lipid classes contained in the total lipidfraction include without limitation one or more of the following: MGDG;MGMG; DGDG; DGMG; non-galactosyl polar lipids including PC and PG;neutral lipids including monacylglycerol, diacylglycerol,triacylglycerol.

Preferably the culture is capable under managed conditions of producinga proportion of its total dry weight as fatty acids; the proportionlying in the range of between 5 and 80%. Preferably the culture iscapable under managed conditions of producing a proportion of its totalfatty acids as EPA; the proportion (by dry weights) lying in the rangeof between 1 and 80%. Preferably the culture is capable under controlledconditions of producing 25% to 60% of total fatty acids as fatty acidscontained in polar lipids; more preferably the proportion is more than30%, more preferably over 40% and even more preferably over 50%.

Preferably the organic carbon component is fed incrementally over time,according to the future predicted growth of the culture in a period of 4to 24 hours.

Preferably the culture is capable under controlled conditions ofproducing 5 to 40% of total fatty acids as fatty acids contained ingalactolipids; more preferably the proportion is more than 10%, morepreferably over 20% and even more preferably over 30%.

Preferably the culture is capable under controlled conditions ofproducing 40 to 70% of EPA as EPA contained in polar lipids (as distinctfrom neutral lipids; more preferably the proportion is more than 50%,more preferably over 60%.

In a second broad aspect the invention provides an EPA-rich compositionderived from a culture as previously described in this section, whereinthe EPA-rich compositions are obtained by a harvesting process includingthe steps of:

-   -   harvesting cells from the culture medium at a selected time;    -   heat or otherwise killing the cells to denature endogenous        enzymes;    -   forming the cells into a cake of biomass;    -   extracting the cake of biomass with a first, non-selective lipid        solvent and recovering the extracted material from the solvent        as a residue; (use of near critical di-methyl ether is one        option)    -   extracting the residue with a second, selective solvent so that        a neutral, EPA-rich lipid composition is separated from the        polar, EPA-rich lipid composition and optionally further        purifying either or both types of EPA-rich compositions in order        to achieve a required standard of purity.

In a related aspect the invention provides an EPA-rich compositionderived from a process as previously described in this section, whereinthe method includes a procedure in which EPA is hydrolysed from a polarEPA-rich lipid class obtained from the polar EPA-rich lipid fractionusing the steps of:

-   -   presenting the polar lipid as a substrate to at least one enzyme        capable of cleaving EPA from the Sn1 position and    -   after cleavage separating the EPA as a free or esterified fatty        acid or metal-salt, thereby obtaining an EPA composition        intended for use as a therapeutic composition or for        prophylactic use.

Preferably the enzymic process uses at least one enzyme selected fromthe range of lipases, phospholipases and galactolipases. Optionally theenzyme is affixed to a surface.

Alternatively the recovery of fractions having a relatively high EPApurity and an increasing yield is accomplished by matching the enzymicrelease of fatty acids over time with a series of recovery steps in acontinuous or multistage batch extraction process.

Alternatively, the addition of materials including (without limitation),an alcohol, hexane or calcium chloride may assist enzymic release byremoving products of the enzyme-catalysed reactions from proximity ofthe enzyme.

Optionally physico-chemical fractionation of the polar lipid fraction,including techniques such as partitioning, chromatographic fractionationand the like, may precede the application of one or more enzymes.

Preferably said galactolipids rich in EPA will be contained inpreparations of the biomass of organisms.

More preferably said galactolipids rich in EPA will be contained in thetotal lipid fraction extracted from the biomass of organisms from whichfraction they may be subsequently separated from the total lipidfraction.

In a first related aspect the invention provides a first method forisolating useful products comprising the steps of: (a) taking apreparation of the biomass of the cultured organisms,

(b) extracting the total lipid fraction from the biomass, and

(c) isolating one or more lipid classes contained in the total lipidfraction including but not limited to one or more of the followingclasses: MGDG; MGMG; DGDG; DGMG; SQDG; non-galactosyl polar lipidsincluding PI, PE, LPC, PC and PG; neutral lipids includingmonacylglycerol, diacylglycerol, triacylglycerol;

Alternatively, the EPA is concentrated in galactolipid classes which arethe preferred substrate of and/or able to be made accessible to anenzyme capable of hydrolyzing one or more acyl bonds and therebyliberating the EPA contained within the galactolipid classes.

In another alternative, EPA is concentrated within the diacylgalactolipid classes.

More preferably EPA is concentrated within themonogalactosyldiacylgalactolipid (MGDG) class.

Preferably the EPA-rich composition is processed after separation inorder to further purify the composition, using known techniques thoughusefully not having to contend with DHA.

Preferably the undesired molecules (even if present) are not cleavedfrom the polar lipid, so that (for example) the EPA composition issubstantially free of DHA which remains acylated to the glycerolbackbone.

Preferred known techniques for separation include low temperaturecrystallisations, purification processes taking advantage ofdifferential solubility of fatty acid esters or salts in varioussolvents including ionic solvents, precipitation using metal salts, theuse of selectively permeable membranes, column chromatography of fattyacids or their esters, supercritical fluid chromatography, urea additioncrystallisation, fractional distillation, preparative HPLC,iodolactonization, and selective re-esterification by enzymes.

In a first alternative aspect, the invention provides a compositionderived from a process as previously described in this section, whereinthe composition comprises 50-60% EPA, less than 5.5% arachidonic acidand substantially no DHA. Preferably the composition comprises 50-60%EPA, less than 4.5% arachidonic acid and substantially no DHA. Morepreferably the composition comprises 50-60% EPA, less than 3.5%arachidonic acid and substantially no DHA. Even more preferably thecomposition comprises 50-60% EPA, less than 2.5% arachidonic acid andsubstantially no DHA In a second alternative aspect, the inventionprovides a composition derived from a process as previously described inthis section, wherein the composition comprises 50-60% EPA, less than5.5% arachidonic acid and less than about 2% DHA. Preferably thecomposition comprises 50-60% EPA, less than 4.5% arachidonic acid andless than about 2% DHA. More preferably the composition comprises 50-60%EPA, less than 3.5% arachidonic acid and less than about 2% DHA. Evenmore preferably the composition comprises 50-60% EPA, less than 2.5%arachidonic acid and less than about 2% DHA

In a third alternative aspect, the invention provides a compositionderived from a process as previously described in this section, whereinthe composition comprises 60-70% EPA, less than 4.5% arachidonic acidand substantially no DHA. Preferably the composition comprises 60-70%EPA, less than 3.5% arachidonic acid and substantially no DHA. Morepreferably the composition comprises 60-70% EPA, less than 2.5%arachidonic acid and substantially no DHA. Even more preferably thecomposition comprises 50-60% EPA, less than 1.5% arachidonic acid andsubstantially no DHA

In a fourth alternative aspect, the invention provides a compositionderived from a process as previously described in this section, whereinthe composition comprises 60-70% EPA, less than 4.5% arachidonic acidand less than about 1.5% DHA. Preferably the composition comprises60-70% EPA, less than 3.5% arachidonic acid and less than about 1.5%DHA. More preferably the composition comprises 60-70% EPA, less than2.5% arachidonic acid and less than about 1.5% DHA. Even more preferablythe composition comprises 50-60% EPA, less than 1.5% arachidonic acidand less than about 1.5% DHA.

In a fifth alternative aspect, the invention provides a compositionderived from a process as previously described in this section, whereinthe composition comprises between 95 and 99% EPA, less than 1% ofarachidonic acid and less than about 0.5% of DHA.

In a sixth alternative aspect, the invention provides a compositionderived from a process as previously described in this section, whereinthe composition comprises between 95 and 99% EPA, less than 0.5% ofarachidonic acid and less than about 0.5% of DHA.

In a seventh alternative aspect, the invention provides a compositionderived from a process as previously described in this section, whereinthe composition comprises between 95 and 99% EPA, less than 1% ofarachidonic acid and less than about 0.1% of DHA.

In an eighth alternative aspect, the invention provides a compositionderived from a process as previously described in this section, whereinthe composition comprises between 95 and 99% EPA, less than 0.5% ofarachidonic acid and less than about 0.1% of DHA.

In a ninth alternative aspect, the invention provides a compositionderived from a process as previously described in this section, whereinthe composition comprises between 99.6 and 99.9% EPA, less than 0.1% ofarachidonic acid and less than 0.1% of DHA.

In a further alternative aspect the invention provides for use of acomposition, as previously described in this section, in the manufactureof a medicament for treatment of a person affected by certain medicalconditions or disorders including but not limited to those selected fromdiabetes (type I, and type II), glycaemic disorders diabetes-associatedhypertension, cancer, osteoarthritis, autoimmune diseases, rheumatoidarthritis, inflammatory and auto-immune diseases other than arthritis,respiratory diseases, neurological disorders, neurodegenerativedisorders (including Huntington's disease, Parkinson's disease,Alzheimer's disease, schizophrenia, major depression, unipolardepression, bipolar depression, obsessive compulsive disorder,borderline personality disorder, post natal depression, organic braindamage, and traumatic brain injury), renal and urinary tract disorders,cardiovascular disorders, cerebrovascular disorders, degenerativediseases of the eye, psychiatric disorders, reproductive disorders,visceral disorders, muscular disorders, metabolic disorders, prostatichypertrophy and prostatitis, impotence and male infertility, mastalgia,male pattern baldness, osteoporosis, dermatological disorders, dyslexiaand other learning disabilities, cancer cachexia, obesity, ulcerativecolitis, Crohn's disease, anorexia nervosa, burns, osteoarthritis,osteoporosis, attention deficit/hyperactivity disorder, and early stagesof colorectal cancer, lung and kidney diseases, and disorders associatedwith abnormal growth and development.

In a third broad aspect the invention provides a composition prepared aspreviously described in this section, wherein the composition isprepared in the form of a human dietary supplement for therapeutic orprophylactic use.

In a first (therapeutic) alternative, the invention provides for use ofa polar lipid, prepared as previously described in this section andincluding an effective amount of EPA in the manufacture of a medicamentfor use in treating the medical conditions or disorders as previouslylisted in this section.

Preferably the polar lipid is formulated in order to provide aprophylactic, health or dietary daily health supplement including anamount in the range of from 0.1 to 50 grams of EPA.

Alternatively the amount is in the range of from 0.5 to 5 grams of EPA.

(Note that even if an EPA-rich polar lipid composition suitable for foodand dietary supplement use has a relatively low content of EPA; it is arelatively high ratio of EPA to undesirable molecules that renders thecomposition fit for purpose, since the undesired molecules exist at asubstantially low level in the composition).

Preferably the content of EPA in the relatively low purity compositionis less than about 20% and the content of undesired molecules remainslow even if the purity of the EPA is below 20%.

In a yet further preferred aspect, the invention provides one or morerelatively low purity EPA compositions which are neverthelesspharmaceutically effective owing to a substantially low level ofundesired molecules in one or more lipid fractions.

More preferably the content of EPA between about 10% and about 80%.

Preferably the availability of such polar lipid compositions broadensthe choice of compositions having a high ratio of EPA to undesirablemolecules which is currently restricted to highly purified EPA from fishoil, which is less accessible to the general population.

More preferably the amount is formulated so as to be suitable forrepeated ingestion as a prophylactic, health or dietary daily healthsupplement.

Preferably the products when consumed are capable of promoting brain andmental health, cognition and behaviour.

Preferably the products when consumed are capable of eliciting healthpromoting effects on any of the following non limiting list of bodysystems and tissues; auditory, appetite, arousal, balance, blood, bone,bowel, cardiovascular, digestive, endocrine, enteric, emotional,gastric, hair, hepatic, immune, lymphatic, kineaesthetic, marrow,memory, metabolic, musculoskeletal, neurotransmitter, nasopharyngeal,pancreatic, musculoskeletal, reproductive, respiratory, ocular,oesophagal, olfactory, palate, pulmonary, proprioceptive, renal, skin,sleep, stomach, sensorimotor, skin, urinogenital, wound healing.

In a further alternative, the prophylactic, health or dietary supplementis formulated as a solid substance compatible with direct ingestion byhumans; the range of formulations including: a cake, a powder, granules,tablets, boluses, pills, capsules, lozenges or beads.

In one option, the EPA is re-esterified or combined with polar lipids,or alternatively the EPA moiety is cleaved from the galactolipids andre-esterified into phospholipid fractions.

More preferably intact or semi-intact polar lipids rich in EPA arecapable of efficiently passing through endothelial and other peripheralcell membranes in mammals.

Preferably said polar lipid fatty acids are incorporated into cellmembranes within the mammal.

A prophylactic, health or dietary supplement as previously described inthis section, wherein the health supplement is provided as a liquidsubstance carrying finely dispersed EPA-rich extracted polar lipidssuitable for consumption as a beverage; the range of beveragesincluding, (without limitation) water, beer, wine, milk, spirits, sportsdrinks, juices and carbonated drinks, and optionally includes at leastone stabilizing substance.

Preferably the EPA-rich extracted polar lipids are encapsulated suchthat n-3 HUFAs and other PUFAs are protected from light includingultraviolet light in order to assist long-term stability.

Preferably the EPA-rich extracted polar lipids are encapsulated suchthat n-3 HUFAs and other PUFAs are protected from oxidative degradation.

Optionally, in the case of beverage preparations, the EPA-rich extractedpolar lipids (including galactolipids) will be released into thebeverage from a temporary encapsulation shortly prior to consumption.

A prophylactic, health or dietary supplement as previously described inthis section, wherein the health supplement is formulated as a substanceselected from the range including: a solution, a suspension, a solidmass, a powder, granules, or the like; the substance including aneffective amount of an extracted galactolipid class rich in EPA and,when in use, is compatible with incorporation into a manufacturedfoodstuff.

An EPA-rich composition suitable for use as an EPA-rich human or animaldietary supplement derived as previously described in this section,wherein the process includes the steps of obtaining the cake of biomassas harvested from the culture, and of preparing substantially the entirebiomass for consumption.

In a yet further alternative, the residual whole cell content isutilized as a product in particular suitable for use as a food formammals including monogastric and ruminant animals and/or aquaculturespecies including finfish and crustaceans; there being valuable residualcompounds e.g.—sulpho galactolipids, carotenoids, other pigments, aminoacids, and other fatty acids capable of serving as foods.

Optionally a method for extracting EPA from a biomass derived fromprocedures whereby certain biomasses produced via the use of in vitrotechniques accumulate commercially useful quantities of galactolipidsrich in EPA at the same time as exhibiting high EPA productivity whengrown under conditions according to those previously described in thissection, in relation to the first broad aspect, may commence by firstremoving undesired fatty acids or lipids with a relatively low EPAcontent, and later extracting the EPA.

In a fourth broad aspect the present invention discloses apharmaceutically pure composition of EPA, wherein the fatty acidcomposition of the composition preferably contains (a) about 80 to 100%EPA and (b) little or no other omega 3 or omega-6 fatty acids; suchcompounds being useful in the treatment of those medical conditions thatrespond to medication with EPA.

Preferably, but not solely, the pharmaceutically pure composition of EPAis derived from galactolipids.

Preferably said pharmaceutical compositions will contain EPA as at least90% of total fatty acids in the composition and may be substantiallypure EPA.

Preferably the content of any one of the fatty acids selected from thenon-limiting range of undesirable

compounds including: DHA, AA, DPA, 18:4 n-3, 18:3 n-3, 18:2 n-6 is lessthan 2% of total fatty acids in the composition and more preferablyapproaches zero.

In a related aspect these EPA compositions will contain very low orundetectable levels of undesirable molecules which are eitherstructurally similar to, or biologically related to, or antagonistic tothe desired effects of EPA when administered to a mammal.

PREFERRED EMBODIMENT

The description of the invention to be provided herein is given purelyby way of example and is not to be taken in any way as limiting thescope or extent of the invention. Throughout this specification unlessthe text requires otherwise, the word “comprise” and variations such as“comprising” or “comprises” will be understood to imply the inclusion ofa stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

Most approaches to culturing single-celled organisms for EPA productionhave been based on solely heterotrophic culture, or else cultures grownunder conditions providing substantial levels of light; substantiallydaylight or artificial equivalents. The present invention includes anovel culture-related aspect, wherein polar lipids rich in EPA areco-produced together with neutral lipids in cultures that are largelyheterotrophic in that they are mixotrophic cultures producing usingsignificantly lower photosynthetically active average irradiances insidethe culture. These will optimally be subphotosynthetic (i.e., less than10 μmol photons m⁻²s⁻¹) but may be as high as 40 μmol photons m⁻²s⁻¹.

The invention exploits the tendency for EPA productivity in polar lipidsto increase and for EPA to become localised within particular polarlipid classes under these conditions. Compositions are provided thatfacilitate subsequent purification of EPA-rich lipid classes, or may bedirectly incorporated into novel supplements rich in EPA.

Galactolipids are incorporated as a major structural component of themembranes of chloroplasts. Other polar lipids including phosphatidylcholine (PC) are also involved in transfer of EPA to the chloroplasts.Certain types of marine algae, but not all, generate intracellularchloroplasts if illuminated and so would be expected to generate thesephotosynthetic lipids in response to light. The invention takesadvantage of the non-linear relationship between provision of light andsubsequent production of photosynthetic lipids.

The inventors are aware that the neutral fraction of lipids will alsocontain significant amounts of EPA. This material is likely to be ofcommercial value in itself and fatty acid derivatives thereof could bepurified by traditional or emerging methods mentioned below to give anEPA product. Thus the present invention should be seen as an adjunct toneutral lipid production rather than a replacement.

Low light levels lie in a range wherein organic carbon consumption isreduced or the efficiency of its use is improved, yet without thetechnical complications of providing high levels of light either by useof outdoors culture with technical and environment-related complicationsor by the use of large amounts of artificial light with associatedenergy costs to be met.

As well as largely heterotrophic cultures grown under conditions of lowlight a further option is the induction of nutrient depletion incultures in order to encourage particular types of polar lipidproduction. Such induced deficiencies include phosphate deficiency whichare expected to shift lipid production from phospho- to galacto-lipids.Silicate depletion in diatoms is also contemplated by the invention as ameans of causing photosynthetic lipids to accumulate.

The invention makes use of the specific molecular structures of certainpolar lipids in certain organisms under certain conditions in order tofacilitate purification of EPA. In association with the above structuralspecificity, the invention contemplates the use of particular enzymeshaving specific appetites such as stereospecificity in order tofacilitate purification. In addition, the unique physiochemicalproperties of photosynthetic polar lipid classes produced, including thepolar nature of the galactolipid molecules, provides useful methods ofadministering EPA.

Finally the invention discloses formulations including the EPA andgalactolipid-rich compositions and applications for the formulations.

Sources of the Cultured Cells

The particular micro-organism that has been used in the proceduresdescribed at the time of filing this specification is Nitzschia laevis.Cells were obtained from the University of Texas Microalgal Collectionwhere they are deposited with the call reference UTEX 2047. We expectthere to be other micro-algae capable of largely heterotrophic growth ofthe “coloured” set (those having photosynthetic machinery) to exhibitcomparable or improved rates of production. At this time, we have notcarried out any strain selection experiments designed to encourageexpression of superior metabolic profiles under an imposed set ofartificial culture conditions, and which allow cells having thoseprofiles to be isolated. We would, however, expect a measurable gain inprocess efficiency (with regard to EPA production) to result.

At this point, trials with (a) other micro-algae, (b) selectedmicro-algae, (see above) or (c) genetically modified micro-algae (see“variations”) have not been done although all such trials are includedin the scope of the invention.

Scaling of Production

Large scale commercial cultures of microalgae can be produced accordingto the methods of this invention under closely monitored and controlledconditions in a vessels with capacities measured in tens or hundreds ofthousands of litres. Changes in conditions and requirements associatedwith the scale of the culture vessel (e.g. cooling/heating, mixing, gasmass transfer) would be anticipated by those skilled in the art.

Further Purification of EPA

Once material with a relatively high level of EPA has been obtained itmay be purified further by a number of means. These include lowtemperature crystallisations, purification processes taking advantage ofdifferential solubility of fatty acid esters or salts in varioussolvents including ionic solvents, precipitation using metal salts, theuse of selectively permeable membranes, column chromatography of fattyacids or their esters, supercritical fluid chromatography, urea additioncrystallisation, fractional distillation, preparative HPLC,iodolactonization, and selective re-esterification by enzymes.

Enzymes and their Provenance.

An “ideal enzyme” for use in the invention would be able to excise sidechains from the glycerol backbone of any polar lipid class if the sidechain comprises a EPA. Whilst several lipases have been isolated thatshow selectivity for chain length we know of no cases where absolutespecificity based on chain length has been demonstrated. None of theseenzymes is currently available for industrial processes.

Many known lipases have the restricted ability of being able to act atthe Sn1 position only, which suggests the production and isolation ofpolar lipids having a desired n-3 HUFA predominantly at the Sn1 positionwould be a route to enrichment. Selection of a particular enzyme for usein a commercial process is also cost-dependent and it may be necessaryto rely on those lipase-type enzymes already produced in bulk for use inthe dairy industry or the baking industry, which includes 1,3 specificlipase-type enzymes made from fungi—for instance thelipase/phospholipase derived from Aspergillus spp, “Bakezyme PH 800 BG”(DSM Food Specialities), or the lipase derived from Rhizopus oryzae,“Piccantase R8000”. The engineered enzyme “Lecitase Ultra” (Novozymes)has 1,3 specific lipase activity but at elevated temperaturesdemonstrates phospholipase A1 activity. Both activities are likely to beof use in the isolation of fatty acids from the Sn1 position of polarlipids.

At this time, attention has been applied in particular tophospholipases. However it will be appreciated that other lipases andgalactolipases are also of relevance in the extraction of EPA and ofEPA-containing materials from biomass. Clearly there is an opportunityfor further exploitation and optimisation of enzyme specificity withinthe invention, for it is likely that enzyme-based purification will havea number of advantages over physico-chemical separation of fatty acids.Tasks such as separation of DHA from DPA or 18:2 from 18:4 fatty acidsare relatively difficult under existing methods many of which arehampered by reliance on physico-chemical factors such as melting pointsor molecular sizes.

The current specification discloses a method to produce a relativelypure EPA composition by exposing a microalgal biomass rich ingalactolipid EPA to an enzyme.

In some versions of the commercial process disclosed herein it is likelythat any enzymes used will be adsorbed on to a surface or otherwiseretained within the process, in order to conserve supplies, by the useof techniques for handling enzymes that are well known in the art. It isalso likely that optimisation of working conditions for a selectedenzyme will provide a significantly improved rate of attack and a morespecific type of attack, as a result of exploitation of workingconditions well known to those skilled in the art, such asconcentration, pH, temperature, presence of salts, or the presence ofcompeting compounds that inhibit undesired modes of action.

In general terms the polar lipids can be either (A) isolated asparticular types, or (B) used as one, collective group. For either A orB, they may then be (i) used directly, (ii) further processed intoEPA-rich fatty acid compositions by cleaving the fatty acids from thelipid species. In the case of further processing, a polar lipid fractionor fractions may be hydrolysed with a specific phospholipase (or otherlipase), the released EPA captured by a suitable acceptor molecule.Examples of acceptors include: glycerol and alcohols including ethanol,propanol, iso-propanol, or long-chain alcohols (which will yield waxes).Alternatively, the EPA may be transferred by a phospholipase,galactolipases, or other lipase on to a suitable carrier type molecule(such as phosphatidylcholine (PC)). Metal salts-membrane separation.

Typical Applications

Typical applications include, for (i): production and use of ultra pureEPA and an active pharmaceutical ingredient (ii) production and use ofnon-pharmaceutical EPA-only therapeutic compositions (iii) productionand use of EPA-rich polar lipid containing foods, functional foods andfood supplements, and (iv) production and use of whole-cell products forfoods or food supplements.

Example 1 Mixotrophic Batch Cultures of N. laevis

Actively growing cells of the species N. laevis obtained as above areproduced in 200 mL of media in stoppered 500 mL Erlenmeyer flasks.Multiple flasks are used to produce large volumes of material. Aninoculum of 0.2 g L⁻¹ of exponential or early stationary phase cells isused. Flasks are incubated in temperature- and light-controlled growthchambers by placing them on orbital shakers at around 200 rpm tomaintain the cells in suspension and aid in gas transfer betweenatmosphere and media. Temperature is maintained at 20° C. Light isprovided at an average irradiance of photosynthetically active light inthe culture of 40 μmol photons m⁻²s⁻¹ as measured by an Apogee quantumsensor digital pyranometer and calculated from conditions such asculture depth and cell density. Aliquots of culture are taken duringgrowth to determine the dry weight of the culture at that time point.Cultures are fed a heat sterilised glucose stock solution (400 g L⁻¹)daily at a level that is projected to provide organic carbonrequirements for the predicted biomass production over the subsequent 24hours. Total glucose added to culture over the entire culture periodamounted to 3 grams per litre.

Culture Medium

Initial concentrations of nutrients in standard media are typically, perlitre:

(a) 50 ml Salt stock solution which comprises NaCl; 160.0 g, MgSO4 7H₂O;44.0 g, KCl; 10.8 g, CaCL2; 2.04 g, KH2PO4; 0.8 g per 1 L of distilledwater.

(b) 50 ml Nitrogen stock solution, which comprises the following: NaNO3;17 g, and yeast extract; 16 g per litre of distilled water.

(c) 10 ml-Tris buffer stock solution. This stock is made by dissolving89.2 g Tris buffer in 1 L distilled water.

(d) 5 ml Trace Metal stock solution, which contains the following, per100 ml; (NH4)6Mo7O24 4H₂O; 0.556 g, CoCl2 6H₂O_(—)0.046 g, MnCl24H₂O_(—)0.500 g, Na2MoO4 2H₂O_(—)0.048 g, H3BO3_(—)61.120 g,ZnCl2_(—)0.622 g, H2SO4 (concentrated); 18 ml.

(e) 2 ml of vitamin solution which is made by dissolving 6 g 0.1%vitamin B12, 0.01 g Biotin and 0.01 g Thiamine in 100 ml distilledwater.

(f) 5 ml Sodium Metasilicate stock solution which is made by dissolving24 g Na2SiO3 in 1 L distilled water).

(g) 2.7 ml of Chelated Iron stock which is made by dissolving 0.81 gFeCl3 6H₂O in 10 ml of 0.1N HCl and dissolving 10 g NaEDTA in 100 ml0.1N NaOH.

(h) 1 mL Copper sulphate stock which is made by dissolving 9.8 mg CuSO₄5H₂O in 1 L distilled water.

Biomass Dry Weight Determination

Biomass dry weight is measured, using the pre-weighed glass fibre filtermethod as follows. A 10 ml sample is removed from a largerrepresentative sample taken whilst stirring to achieve a broadlyhomogenous dispersion of cells and cell aggregates; culture flasks aregenerally sterilised with a Teflon-coated magnetic stir bar in place toaid with this. The 10 ml sample is placed in a centrifuge tube and spunat 3000 rpm in a Heraeus Sepatech Megafuge 1.0 with swing-out rotor for4 min and the liquid decanted leaving a cell pellet. The cell pellet iswashed with phosphate-buffered saline and re-centrifuged. A Sartoriusglass fibre filter is washed by passing 1 litre of deionised waterthrough the filter then dried overnight in a vacuum oven at 30° C. priorto being weighed. The 10 ml sample is passed through the preweighedfilter in a vacuum filter apparatus and is then placed in an oven at 60deg C. for two hours prior to being reweighed. The difference in gramsbetween the pre and post weights times 100 is taken as a measure of thedry weight per litre.

Harvesting and Extraction of Lipid-Containing Material.

Cells are harvested after 3 days of growth since at this point theculture(s) are still in exponential phase. Cellular extract containingthe lipids can be obtained by Folch extraction following the method ofBligh and Dyer (1959). Cells from several flasks are combined to allowproduction of sufficient material for further use.

Total Fatty Acid Analysis.

Total fatty acid analyses of samples of cellular extract are obtained toidentify the composition of the cultured material. Addition of aninternal standard such as C23:0 to the reaction allows measurement ofthe total fatty acid content of the cells. The method of fatty acidproduction entails a basic transesterification with 0.5M methoxide inmethanol followed by an acidic transesterification using dry HCl inmethanol. Fatty acid methyl esters are recovered by extracting withhexane and drying with sodium sulphate before analysis using gaschromatography. The sample is run on a 30 m×0.25 mm ID Framewax(crossbond polyethylene glycol) glass capillary column contained withina Shimadzu 2010 GC by autoinjection. By ramping the column temperaturefrom 145 to 240° C. over the course of 50 minutes and then leaving thecolumn at 240° C. for a further 10 minutes fatty acids is identified byco-chromatography with known standards supplied by Restek.

Lipid Separation.

Cellular extract is separated into polar and non-polar fractions usingcolumn chromatography. 0.5-2 g of the cellular extract is dissolved in asmall volume of diethyl ether and loaded onto a column containing 40 gsilica gel (with a mesh size of 230-350) in diethyl ether. 10 mL ofdiethyl ether and 80 mL of chloroform is used to elute non-polarmaterial including the triglycerides. Further addition of 10 mLchloroform:methanol (1:1 v/v) and 80 mL methanol to the column elutesthe polar material including galactolipids and phospholipids. These twoclasses of material are collected separately and dried down beforesamples are subjected to fatty acid analysis similar to that describedabove. The polar fraction is then further separated by placing it on asecond chromatographic column. A column is constructed of silica gel inchloroform and is washed with successive washes of 2 column volumes eachof 99:1 (v/v), 49:1 (v/v), 29:1 (v/v), 19:1 (v/v), and 9:1 (v/v)chloroform:methanol and 2 column volumes of methanol. Further steps areadded as required to separate other galactolipids and phospholipids fromone another if so desired.

Lipase Based Separation of Fatty Acids from Specific Positions withinLipid Molecules.

The 1,3 specific lipase and phospholipase A1 “Lecitase Ultra®” fromNovozymes is used to cleave the fatty acids from the Sn-1 position ofMGDG isolated in the manner described above. 5 mg of MGDG is dissolvedin 3 mL of methanol whilst 12 u of enzyme is dissolved in 3 mL 10 mMcitric acid buffer at pH6.0. These are incubated together at 60° C. for5-15 minutes and after incubation the reaction could is washed 3 timeswith 2 mL hexane to collect the free fatty acids produced. The hexanewashes are collected in a fresh tube with 3 mL methanol and the mixtureincubated at 50° C. for 2 hours to produce Fatty acid methyl esters. Atthe end of this period the hexane layer is removed and concentratedbefore being analysed on a GC.

Results.

After 72 hours a biomass dry weight reaches 3 grams per litre in flaskculture. Fatty acids form at least 8% of the dry material grown in theculture. EPA reaches 24% of total fatty acids.

Cultures of Nitzschia laevis grown in this manner demonstrate doublingtimes as low as 12 hours.

The analysis of the fractions recovered from the first chromatographiccolumn shows that roughly equal amounts of fatty acids are recovered inpolar and non-polar lipid fractions. 67% of the EPA is located in thepolar fraction.

Analysis of the fractions by GC shows that around one third of the polarfatty acids elute in the 9:1 (v/v) fraction, Thin Layer Chromatographyof the fractions indicates that the 9:1 (v/v) fraction contains the bulkof the galactolipid MGDG with the remainder of the MGDG and all otherlipid classes eluting with methanol.

Table 1 (below) shows in the left hand column the total fatty acidprofile of MGDG isolated using the method described in the presentexample and in the right hand column the total fatty acid profile offatty acids recovered from the hydrosylate.

TABLE 1 Total and enzyme-hydrolysed fatty acids from MGDG. Figures arepercentage of total fatty acids loaded on the GC. Please note that whereresults are expressed as “ND” (“not detected”) the amount present wasbeyond the limits of detection of our instrument. Total Fatty acidEnzyme-liberated production from Fatty acids from Fatty acid Name MGDGMGDG C14:0 2.44 3.81 C16:0 3.94 2.17 C16:1 c9 35.79 27.06 C16:2 4.071.62 C16:3 14.15 0.49 C16:4 0.47 ND C18:2 c9,12 0.26 0.14 C18:2 t9,120.04 ND C18:3 c9,12,15 0.48 0.89 C18:3 c6,9,12 0.22 ND C18:4 n3 0.880.43 C20:2 c11,14 0.09 0.76 C20:4 c5,8,11,14-AA 2.99 5.32 C20:4c8,11,14,17 0.19 0.23 C20:3 c8,11,14 0.05 ND C20:5 c5,8,11,14,17-EPA30.11 54.93 C22:2 c13,16 0.07 0.64 C22:5-DPA 0.2 0.21 C22:6-DHA 0.68 NDUnknown molecules 1.75 0.21 Other saturates 0.47 0.48 Othermonounsaturates 0.68 0.61

Discussion

The method allows significant increases in the polar lipid production ofthe organism to take place over that which is measured in a purelyheterotrophic culture (where 75% or more of the fatty acids are seen inthe non-polar fraction).

Roughly one third of the polar fatty acid in the example is contained inthe MGDG fraction as compared with only around 15% in purelyheterotrophic cultures.

Application of the enzyme in the present example provides for enrichmentof EPA and the exclusion of DHA from the fatty acids recovered from thehydrosylate as compared to the total fatty acids of the MGDG fraction.

Whilst only 30-35% of the total MGDG fatty acids are recovered in thehydrosylate in the present example 57.0% of the EPA in the sample isrecovered by the enzymatic process confirming predominance in the Sn1position of MGDG.

Example 2 Largely Heterotrophic Batch Cultures of N. laevis

Flask cultures of N. laevis is produced according to the method ofexample one except that: 5-10 grams of glucose is added over the courseof the culture run. Light is provided at an average irradiance ofphotosynthetically active light in the culture of 10 μmol photonsm⁻²s⁻¹. Harvesting, separation and analysis techniques are all accordingto the method of example one.

Results

After 72 hours a biomass dry weight reaches between 3 and 5 grams perlitre in flask culture. Fatty acids form at least 10% of the drymaterial grown in the culture. EPA reaches at least 20% of total fattyacids. Analysis of the fractions obtained from the first chromatographiccolumn shows that 35 to 40% of fatty acids are recovered in the polarfraction and 60-65% in the non-polar lipid fraction. EPA ispreferentially located in the polar fraction.

Analysis of the fractions by GC shows that between 30 and 40% of thepolar fatty acids elute in the 9:1 (v/v) fraction. Thin LayerChromatography of the fractions indicates that the 9:1 (v/v) fractioncontains the bulk of the galactolipid MGDG with the remainder of theMGDG and all other lipid classes eluting with methanol.

Analysis of the MGDG fraction shows that over 30% of the fatty acids areEPA. Of the material recovered from enzymatic hydrolysis, between 50 and60% is EPA.

Discussion

More fatty acids were recovered in the non-polar fraction than the polarfraction but the amounts of fatty acids in the polar fraction are stillsubstantially higher (50-200% greater) than those from a similarheterotrophic culture grown in the absence of light. EPA is found as ahigher proportion of fatty acids in the polar fraction both incomparison to the non-polar fraction and in comparison to a polarfraction from a totally heterotrophic culture.

Significantly, although the amount of lipids recovered in the polarclass is lower as a proportion of the whole when compared to mixotrophicor phototrophic growth, the improved growth rates under largelyheterotrophic conditions means that polar and galactolipid yields areequivalent to or better than those of example 1.

Example 3 “Perfusion Culture” Mode of Culturing N. laevis IncludingVariation of Nutrient(s) and/or Exposure to Sub-Photosynthetic LightIntensities

Larger volumes of actively growing cells of the species N. laevis aregrown under closely monitored and controlled conditions in a 20 litrevessel, having an effective working capacity of 18.5 litres. The vesselis internally lined with “Teflon®” and comprise a stirred, jacketedtank. The jacket is provided with hot or cold water as required in orderto maintain an internal temperature of 20° C., as sensed by internalprobes and controlled with a SCADA device controlling water valves. Amechanical seal admits an impeller shaft of 19 mm diameter, having a6-blade Rushton impeller at one end, placed near an air sparger, and amarine impellor 250 mm from the end. A 0.25 kW 3-phase 6-pole motordrives the shaft at between 100 and 900 revolutions per minute. Motorspeed is controlled with a variable speed drive capable of receiving ananalogue signal from the supervisory control device. Pressurised air(1.5 bar) is injected through a sterilizing filter at a rate of between2 to 10 litres per minute to the air sparger. The air flow is measuredwith a Dwyer flow meter model TF 2110 and the flow rate is controlledeither manually through the use of a regulator or by a “Festo”proportional solenoid controller. Similarly gas outflow from the vesselis measured and regulated. Dissolved oxygen is measured by an oxygensensor (Broadley-James Corporation, Irvine, Calif.) and the dissolvedoxygen maintained at around 50% of saturation via supervisory controlfeedback loops controlling motor speed and, to a lesser degree, airflow.

The pH of the culture is maintained at pH=8.5 (or at another pH ifdesired) by using an immersed pH sensor (Broadley-James Corporation,Irvine, Calif.) in a closed control loop, driving a peristaltic pump forthe addition of either alkali (as NaOH or KOH) or for the addition ofacid (as HCl or Acetic acid) as required.

Concentrations of nutrients, including feed stocks that are sources ofnitrogen, silicate, phosphorus, glucose, and organic carbon (e.g.glucose) are separately controlled by feeding sterile stock solutions ofdesired concentrations aseptically through corresponding peristalticpumps. Sterile basal media is also supplied to the vessel through anaseptic pump. Culture volume is monitored using a Kubler level sensorand input of basal media or nutrient controlled by the SCADA device.

Precautions related to sterility include operations being conducted in aproduction environment equipped with an air lock and supplied withfiltered sterile air to create a positive pressure directing air awayfrom culture vessels. Staff follow protocols well known to those skilledin the art designed to minimize any accidental introduction ofcontaminants into the production environment. All stocks that canwithstand heating are autoclaved at 121° C. for 15 minutes or 132° C.for 4 minutes. Remaining media is filtered through 0.2 micron filters.All pumps, lines and vessels are steam sterilized prior to use of theapparatus and all exposure of culture or cells to the externalproduction environment is undertaken in a laminar flow cabinet inaccordance with sterile technique.

The vessel is inoculated by introducing a freshly growing culturethrough a previously steam-sterilised manifold in order to achieve aninitial concentration of from 0.1 to 1 g per litre of cells in thevessel. Motive pressure for the transfer is provided throughdisplacement of the inoculating culture with sterile air.

An optical density probe is also be immersed in the tank in order toindicate the amount of biomass present in a culture. The culture vesselis also be provided with one or more settling devices, which areexternal separating funnels into which cells within their medium may bepumped from time to time aseptically via the operation of a peristalticpump. These devices function by allowing cell-containing media to bepumped into them to settle whilst at the same time permitting spentmedia, substantially free of cells to be removed in a sterile manner.The suspension rich in settled cells located at the bottom of thesettling device is then pumped back into the culture vessel via thealternate operation of a second peristaltic pump. The reduction in totalmedium volume in the main culture vessel is made up with fresh media, sothat excretory products can be taken out of the vessel and freshnutrients added. The dimensions and volume of the settlers is optimisedvia deign methodologies well know to those skilled in the art to allowthe total volume of the tank to be changed over a 24 hour period via theoperation of one or more settlers whilst minimizing the residence timeof cells in the settlers.

Means for providing light to the micro-algal cells during cultureinclude one or more of: use of an optically translucent or transparentsection of the vessel with a surface illuminated externally, insertionof light guides through ports from the exterior into the culture,insertion of a sterilisable light emitting device (e.g. fibre optics,conventional bulbs or LEDs). Cells can be pumped of cells, using aperistaltic pump or the like, from within the culture medium outside andalong tubes that are exposed to a light source. For instance atransparent flat panel vessel illuminated using artificial light isused. Relative amounts of time spent in the main vessel and the flatpanel system determine the amount of light that the cells are exposedto.

A low continuous level of exposure using, for example, light guides maybe preferable to a high intermittent exposure in an external system.Alternatively cells may be pulsed intermittently with high intensitylight so as to achieve an average low intensity over the period of theculture.

A preferred method for the production of Nitzschia laevis is the use oflow intensity light providing a photosynthetically active averageirradiance in the culture of less than 40 micromol photons per squaremeter per second,

An even more preferred method for the production of Nitzschia laevis isthe use of low intensity light providing a photosynthetically activeaverage irradiance in the culture of 1-10 micromol photons per squaremeter per second.

The biomass is harvested as a batch after 5 to 9 days of growth andsubjected to extraction with supercritical dimethyl ether (DME). In thisprocess cells are first collected and heat killed at 70° C. for 15minutes to denature endogenous enzymes. The cells are then spray driedto form a powder with less than 10% water content. Supercritical DME (at60° C. and 40 bar pressure) is then used to extract material from thepowder and recovered. Removal of the DME leaves a tar-like extract whichcontains the lipids as well as pigments and other cellular material.Around 50% of the dry weight is extracted using this method. Subsequentextraction of the complex lipid mixture with supercritical CO₂ may alsobe performed which has the effect of separating polar material (left asa residue in the CO₂ process) from non-polar (dissolved in thesupercritical CO₂). A variety of other methods of isolating lipids areknown to those skilled in the art. The total extract or isolated neutralor polar lipid fraction may be used in its own right or fatty acidsrecovered by direct saponification via methods well known to thoseskilled in the art. Further chromatographic processes as described inexample one can then be utilised as necessary to further purify lipidclasses or fatty acid fractions.

Isolation of fatty acids from the Sn1 position of specific polar lipidclasses is carried out by dissolving the lipid material in methanol andpassing it through a column of immobilised Lecitase Ultra. Addition ofhexane to the material flowing from the column isolates the fatty acidshydrolysed by the enzyme. These are then purified further as desired.Productivity of this system is between 5 and 50 mg EPA per litre perhour.

Variations Transgenic Organisms

The invention may rely on use of higher plants cells normal ortransgenic organisms including but not limited to algae, fungi, andbacteria.

Culture Conditions

A possible improvement option involves growing micro-organisms for aperiod under conditions in which the media is depleted of eithersilicate or phosphate or both in order to cause the organisms to producemore polar galactolipids in their lipid membranes, which aresubsequently extracted. Preferably the nutrient limitation is imposed onthe culture over the last phase of growth prior to harvesting.

Cooling of Cultures

The culture may be maintained under temperatures below the previouslystated 20 degrees Celsius, and above the freezing point of the culturemedium.

One option based on the postulate (whether it is adequate or not) thatEPA serves to render lipid membranes more fluid involves growing themicro-organisms for a period under conditions in which themicro-organisms are cooled; perhaps as far as the freezing point of seawater (−1.8 deg C.) in order to cause the organisms to include more EPAeither as lipids or as galactolipids in their lipid membranes, which aresubsequently extracted. Preferably the cooling is applied to the cultureover the last phase before harvesting.

Uses of Extracted Material Therapeutic Compositions

There is no reason why a therapeutic composition containing less than(for example) 20% EPA yet having substantial absence of otherpotentially antagonistic molecules such as DHA AA etc. should not bejust as effective as a 100% pure EPA oil (not counting esters). Thedrive to get substantially 100% purity could be re-expressed as a desireto have substantially none of the “undesired molecules” such as DHA.Therefore, acceptance of (for example a 10 to 95% pure EPA) becomes amatter of satisfying the relevant regulatory authorities. The role ofthe inventors becomes a matter of exclusion of certain impurities.Further, a therapeutic composition that delivers EPA in a relativelywater-soluble form (or a stable emulsion in water) has substantialformulation advantages.

EPA-Rich Foods Including Galactolipids

A basis for making foods, food supplements, nutraceuticals, ortherapeutic preparations that rely on the EPA held in polar lipidmolecules is that on oral administration of polar lipids rich in EPA toa mammal leads to a significant proportion of their fatty acids beingabsorbed into the blood stream via enteric lymph vessels therebybypassing first-pass liver metabolism and thus providing greaterbioavailability of the EPA.

In addition the polar nature of the lipids renders this form of EPA,which does not behave as in the same manner as a fatty acid or ester oras a neutral lipid, easier to formulate and to administer. Polar lipidsderived from microorganisms would not carry a fishy flavour of the typeusually present in fish oil extracts. Galactolipids and certainphospholipids according to the invention are recognised to be ofparticular utility due to a combination of their high EPA content, lowcontent of other potentially antagonistic molecules and undesirablefatty acids.

The unique physiochemical properties of galactolipids conferred by thehydrophilicity of the polar carbohydrate head group; rendering themexcellent surfactants. This latter property will allow the production ofa range of EPA-only food and in particular EPA-only beverages due to theability of the galactolipids to be dispersed as micelles and remainstable over long periods of time in aqueous oil in water solutions. Invitro techniques can accumulate commercially useful quantities of polarlipids and especially galactolipids rich in EPA at the same time asexhibiting high EPA productivity when grown under conditions accordingto this invention,

An EPA-rich galactolipid that has been manufactured from a cultureaccording to the invention may be prepared for storage, shipping andsale as a substance having one of a variety of physical forms such as asolution a suspension (feasible with water), or in a cake, a powder,granules, tablets, boluses, pills, capsules, or beads. In, for example,a powder, the galactolipid may be bound to inert particles (such as ofstarch) or encapsulated by means well known to those skilled in therelevant arts.

Means to restrict oxidation of the EPA may be included in packaging suchas by sparging with nitrogen. In addition microencapsulation for foodsmay be assisted with phospholipids (such as crude or purified lecithins)so that the EPA is protected The composition includes an effectiveamount of an extracted galactolipid rich in EPA and is suitable for oralingestion either directly or after a technical process of foodpreparation.

Beverages.

In order to deliver a recommended daily intake to a population, abeverage may be a most preferred route since beverages are consumed bymost people. Due to the vulnerability of n-3 HUFA and other PUFAs tooxidative degradation it may be preferable to encapsulate thegalactolipid such that it is protected from light especially sunlight,and released into the beverage shortly prior to consumption.Alternatively it may be desirable to sparge the liquid containing thegalactolipid with an inert gas to prevent exposure of the EPA to oxygen.Carbon dioxide in carbonated beverages may assist dispersion of thegalactolipid. Micelles may also hold dissolved carbon dioxide in thecase of carbonated beverages and the composition of the micelle may bealtered in order to enhance this property. In certain cases it may bepreferable to add glycerol to these beverage preparations to optimisethe dispersion of the galactolipids and also to provide a more desirablemouth feel for the beverage. Alternatively a water-free concentratewhich would be made up by the user with water at or near the time ofconsumption may be distributed. Steps to minimise oxidation of the n-3HUFA within the concentrate would have to be taken including the use oflight-tight packaging, and microencapsulation of the galactolipid.

Milk is a fairly universally consumed beverage and there are manyprocessed variants of “plain” milk on sale. EPA-supplemented milk ismade by adding an EPA-rich galactolipid during processing. The usualpractice is to homogenize and pasteurize milk in a single process. Inorder to minimise exposure of the EPA to heat, the EPA-rich galactolipidis preferably added during or after cooling of the milk.

Spreads.

Spreads may have EPA-rich materials included. Such spreads include theprotein-rich type such as yeast extracts, or fat-based spreads such asaioli, butter and margarine, in which water is the dispersed phase. Inmanufacture, the galactolipid is added either to the fatty component orto the water component and the polar nature of the molecule assists insolubility. Spreads also include jams and jellies. The EPA-richgalactolipid may be added to a jam in the form of an emulsion. Moresolid preparations include sweets and chocolates. The EPA-richgalactolipid may be added as a fat-soluble component during manufacturepreferably after heating in order that the galactolipid is not exposedto heat. EPA-rich solid foods.

EPA-rich galactolipids may be added to ice cream, for example, alongwith selected long-chain fatty acid molecules and optionally aphosphatidyl choline (PC) molecule as a carrier. An alternative is totransfer the EPA on to a selected PC molecule using a selectivegalactolipase/phospholipase. Long-chain lyso-phosphatidylcholine is apossible suitable receptor having advantages in processing and inproducts. The product formed may be insoluble within the enzyme systemwhich tends to drive the equation towards its formation. AlternativelyEPA-rich phosphatidylcholine isolated from the culture may be used.

Whole-cell preparations including effective amounts of EPA.

Whole-cell preparations, which may be intact, partially hydrolysed, orlysed, may be incorporated directly into spread-type foods, bakingproducts, processed meats or other food supplements either (i) as is, or(ii) after homogenisation (by shear or pressure) or (iii) controlledenzymatic or chemical hydrolysis to aid proteolysis. The whole cell willoffer useful protein and generally high HUFA levels, even if somestripping of EPA has taken place.

ADVANTAGES AND INDUSTRIAL APPLICABILITY

The invention as described herein offers:

-   -   1. An industrially upwardly scalable process capable of creating        a compound (such as EPA) that is difficult to synthesise, and so        at the present time is obtained mainly from the limited        resource, marine fish oil, yet is in increasing demand on        account of rising populations and better awareness of the        consequences of inadequate intake.    -   2. The preferred light levels (of up to about 80 μmol photons        m⁻²s⁻¹ if at a steady rate, or at a higher yet equivalent rate        if intermittently provided) are cheaper to provide by artificial        means than the level of full sunlight, or to provide if daylight        is used    -   3. A process as above for providing EPA in a variety of purities        (suitable for therapeutic use) and importantly, the product        includes only traces of the undesired molecule DHA.    -   4. A culture process for creating EPA from a largely        heterotrophic organism that is relatively economical to manage,        nourish and supply with energy in a large-scale manufacturing        environment.    -   5. A process as above for co-producing EPA with triglycerides,        and within polar lipid compounds (for example, galactolipids)        that open up a number of possibilities for therapeutic and        prophylactic administration.    -   6. Acceptable and readily ingested formulations containing        useful amounts of EPA (in terms of nutritional and/or        therapeutic requirements) within polar lipids.

Finally, it will be understood that the scope of this invention asdescribed by way of example and/or illustrated herein is not limited tothe specified embodiments. Where in the foregoing description, referencehas been made to specific components or integers of the invention havingknown equivalents, then such equivalents are included as if individuallyset forth. Those of skill will appreciate that various modifications,additions, known equivalents, and substitutions are possible withoutdeparting from the scope and spirit of the invention as set forth in thefollowing claims.

1. A composition comprising 50-60% EPA, less than 5.5% arachidonic acidand substantially no DHA.
 2. A composition comprising 60-70% EPA, lessthan 4.5% arachidonic acid and substantially no DHA.
 3. A compositioncomprising between 95 and 99% EPA, less than 1% of arachidonic acid andless than 0.1% of DHA.
 4. A composition comprising between 99.6 and99.7% EPA, less than 0.1% of arachidonic acid and less than 0.1% of DHA.